U.S. patent application number 16/096160 was filed with the patent office on 2021-07-22 for lipophosphonoxins of second generation, and their use.
This patent application is currently assigned to USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I.. The applicant listed for this patent is MIKROBIOLOGICKY USTAV AV CR, V.V.I., TRIOS, SPOL. S R. O., USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I.. Invention is credited to Milan KOLAR, Libor KRASNY, Tomas LATAL, Radek POHL, DOMINIK REJMAN, Eva ZBORNIKOVA.
Application Number | 20210220383 16/096160 |
Document ID | / |
Family ID | 1000005509391 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210220383 |
Kind Code |
A1 |
REJMAN; DOMINIK ; et
al. |
July 22, 2021 |
LIPOPHOSPHONOXINS OF SECOND GENERATION, AND THEIR USE
Abstract
Lipophosphonoxins of general Formula I are described, in which
R.sub.1 is C8-C22, preferably C10-C18 and more preferably C12-C16
alkyl, or hexadecyloxypropyl, tetradecyloxypropyl,
tetradecyloxyethyl or hexadecyloxyethyl, R.sub.2 is uracil,
thymine, or cytosine, and R.sub.3 is selected from a group which
contains compounds of general formulas II and III.
Inventors: |
REJMAN; DOMINIK; (Praha 7,
CZ) ; POHL; Radek; (Uholicky, CZ) ;
ZBORNIKOVA; Eva; (Libcice nad Vltavou, CZ) ; KRASNY;
Libor; (Roztoky u Prahy, CZ) ; LATAL; Tomas;
(Hlusovice, CZ) ; KOLAR; Milan; (Olomouc,
CZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
USTAV ORGANICKE CHEMIE A BIOCHEMIE AV CR, V.V.I.
MIKROBIOLOGICKY USTAV AV CR, V.V.I.
TRIOS, SPOL. S R. O. |
Praha 6
Praha 4
Praha 4 |
|
CZ
CZ
CZ |
|
|
Assignee: |
USTAV ORGANICKE CHEMIE A BIOCHEMIE
AV CR, V.V.I.
Praha 6
CZ
MIKROBIOLOGICKY USTAV AV CR, V.V.I.
Praha 4
CZ
TRIOS, SPOL. S R. O.
Praha 4
CZ
|
Family ID: |
1000005509391 |
Appl. No.: |
16/096160 |
Filed: |
April 19, 2017 |
PCT Filed: |
April 19, 2017 |
PCT NO: |
PCT/CZ2017/050017 |
371 Date: |
October 24, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 19/10 20130101;
A61K 31/7072 20130101; A61P 31/04 20180101; A01N 57/24
20130101 |
International
Class: |
A61K 31/7072 20060101
A61K031/7072; C07H 19/10 20060101 C07H019/10; A01N 57/24 20060101
A01N057/24; A61P 31/04 20060101 A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 2016 |
CZ |
PV 2016-243 |
Claims
1: Lipophosphonoxins of general formula I, ##STR00013## wherein
R.sub.1 is selected from C8-C22 alkyl, hexadecyloxypropyl,
tetradecyloxypropyl, tetradecyloxyetyl, hexadecyloxyetyl; R.sub.2
is selected from uracil, thymine, cytosine; and R.sub.3 is selected
from the group consisting of compounds of formulas II-V:
##STR00014## wherein R.sub.4 is H, CH.sub.2NH.sub.2 or CH.sub.2OH,
R.sub.5 is H, NH.sub.2 or OH, R.sub.6 is H, NH.sub.2 or OH, R.sub.7
is H, CH.sub.2NH.sub.2 or CH.sub.2OH, R.sub.8 is H,
CH.sub.2NH.sub.2 or CH.sub.2OH, R.sub.9 is H, NH.sub.2 or OH,
R.sub.10 is H, NH.sub.2 or OH, R.sub.11 is H, NH.sub.2 or OH,
R.sub.12 is H, CH.sub.2NH.sub.2 or CH.sub.2OH, R.sub.13 is NH.sub.2
or NH--CH(NH.sub.2)NH, R.sub.14 is NH.sub.2 or NH--CH(NH.sub.2)NH,
R.sub.15 is NH.sub.2 or NH--CH(NH.sub.2)NH, R.sub.16 is NH.sub.2 or
NH--CH(NH.sub.2)NH, whereas at least one of R.sub.5 and R.sub.6
groups must be NH.sub.2 or at least one of R.sub.4 and R.sub.7
groups must be CH.sub.2NH.sub.2, and whereas at least one of
R.sub.9, R.sub.10 and R.sub.11 groups must be NH.sub.2 or at least
one of R.sub.8 and R.sub.12 groups must be CH.sub.2NH.sub.2; and
their pharmaceutically acceptable salts and/or hydrates.
2: Lipophosphonoxins of general formula I according to claim 1, or
pharmaceutically acceptable salts and/or hydrates and/or mixtures
of such compounds, for use as a medicament.
3: Lipophosphonoxins of general formula I according to claim 1 or
their diastereomers, or pharmaceutically acceptable salts and/or
hydrates and/or mixtures of such compounds, for use as an
antibacterial agent.
4: Antibacterial drug, characterized in that it contains at least
one lipophosphonoxin of general formula I according to claim 1, or
a diastereomer, or a pharmaceutically acceptable salt and/or
hydrate, and/or a mixture of such compounds as the active
ingredient.
5: Disinfectant for other than therapeutic purposes and/or
selective culture medium characterized in that it contains at least
one lipophosphonoxin of general formula I according to claim 1, or
its diastereomer, or a pharmaceutically acceptable salt and/or
hydrate, and/or mixture of such compounds, as the active
ingredient.
6: A method of treatment of bacterial infections comprising the
step of administering the lipophosphonoxins of Formula I according
to claim 1, or their diastereomers, or pharmaceutically acceptable
salts and hydrates, and/or mixtures of such compounds to the
subject in need of such treatment.
7: A method of preparation of a disinfectant comprising the step of
providing lipophosphonoxins of Formula I according to claim 1, or
their diastereomers, or pharmaceutically acceptable salts and/or
hydrates, and/or mixtures of such compounds.
8: A method of preparation of selective cultivation medium for in
vitro cultures comprising the step of providing lipophosphonoxins
of Formula I according to claim 1, or their diastereomers, or
pharmaceutically acceptable salts and/or hydrates, and/or mixtures
of such compounds.
Description
FIELD OF ART
[0001] The invention relates to new substances with antibacterial
effects and their use in vitro and in vivo.
BACKGROUND ART
[0002] Currently, an increasing number of bacteria are becoming
resistant to conventional medicines and new drugs are therefore
needed for treatment of diseases caused by these resistant bacteria
(Davies D., Davies J., Microbiol. Mol. Biol. Rev. 2010, 74(3), 417;
Kesselheim A. S., Outterson K., Health Aff 2010, 29, 1689).
[0003] Recently, lipophosphonoxins of first generation were
reported, exhibiting activity against gram-positive bacteria (J.
Med. Chem. 2011, 54(22), 7884-7898, CZ PV 2011-312, EP2527351).
Furthermore, the mechanism of their effect was described,
consisting of selective disruption of the bacterial membrane (PLoS
One 2015, 10(12), e0145918).
[0004] Lipophosphonoxins (LPPO) are bactericidal substances with
fast kinetics and they are not genotoxic. Maximum tolerated dose
(MTD) in mice after oral administration is very high (>2000
mg/kg) and the bacteria are not able to develop resistance.
Lipophosphonoxins are chemically stable over a broad pH range and
do not pass through a monolayer of CACO-2 cells, which means that
very likely, they will not be absorbed after oral administration.
LPPO belong to the growing family of antibacterial peptidomimetics,
such as cationic steroidal antibiotics (Ferns Microbiol Lett. 2002,
217(1):1-7; Bba-Biomembranes 2007, 1768(10), 2500-2509; J. Med.
Chem. 2002 45(3), 663-669), lipophilic derivatives of norspermidine
(J. Med. Chem. 2014, 57(22), 9409-9423), arylamide foldamers
(Antimicrob Agents Ch. 2011, 55(11), 5043-5053; Angew. Chem. Int.
Edit. 2004, 43(9), 1158-1162) or a promising synthetic bactericidal
antimicrobial peptide LTX-109 (Angew Chem Int Edit 43:1158-62.
Antimicrob Agents Ch 55:5043-53) which degrades the membranes of
harmful microorganisms. These compounds are structurally
heterogeneous; however, they are all amphiphilic molecules
containing a lipophilic portion and a hydrophilic portion, usually
carrying a positive charge. Lipophosphonoxins also share this
structural motif; however their main advantage lies in their
modular structure, allowing systematic tuning of their biological
properties.
DISCLOSURE OF THE INVENTION
[0005] This invention discloses novel compounds of Formula I, which
exhibit strong antibacterial activity against gram-positive and
gram-negative bacteria. In addition to their easy preparation, the
advantage of these compounds is their modular structure which
allows further tuning of their biological properties.
[0006] The invention involves lipophosphonoxins of second
generation of general formula I,
##STR00001##
wherein: R.sub.1 is selected from C8-C22 alkyl (preferably C10-C18
alkyl and more preferably C12-C16 alkyl), hexadecyloxypropyl,
tetradecyloxypropyl, tetradecyloxyetyl, hexadecyloxyetyl; R.sub.2
is selected from uracil, thymine, cytosine; and R.sub.3 is selected
from the group consisting of compounds of general formulas II to
V:
##STR00002##
wherein R.sub.4 is H, CH.sub.2NH.sub.2 or CH.sub.2OH, [0007]
R.sub.5 is H, NH.sub.2 or OH, [0008] R.sub.6 is H, NH.sub.2 or OH,
[0009] R.sub.7 is H, CH.sub.2NH.sub.2 or CH.sub.2OH, [0010] whereas
at least one of the groups R.sub.5 and R.sub.6 must be NH.sub.2 or
at least one of the groups R.sub.4 and R.sub.7 must be
CH.sub.2NH.sub.2; [0011] R.sub.8 is H, CH.sub.2NH.sub.2 or
CH.sub.2OH, [0012] R.sub.9 is H, NH.sub.2 or OH, [0013] R.sub.10 is
H, NH.sub.2 or OH, [0014] R.sub.11 is H, NH.sub.2 or OH, [0015]
R.sub.12 is H, CH.sub.2NH.sub.2 or CH.sub.2OH, [0016] whereas at
least one of the groups R.sub.9, R.sub.10 and R.sub.11 must be
NH.sub.2 or at least one of the groups R.sub.8 and R.sub.12 must be
CH.sub.2NH.sub.2; [0017] R.sub.13 is NH.sub.2 or
NH--CH(NH.sub.2)NH, [0018] R.sub.14 is NH.sub.2 or
NH--CH(NH.sub.2)NH, [0019] R.sub.15 is NH.sub.2 or
NH--CH(NH.sub.2)NH, [0020] R.sub.16 is NH.sub.2 or
NH--CH(NH.sub.2)NH; and their pharmaceutically acceptable salts
and/or hydrates.
[0021] The pharmaceutically acceptable salts include salts with
inorganic or organic anions and particularly, but not exclusively,
pharmaceutically acceptable salts suitable for physiological
administration.
[0022] Pharmaceutically acceptable salts may be salts derived from
inorganic or organic acids. A person skilled in the art will be
able to determine which are pharmaceutically acceptable salts;
particularly they are salts having one or more desirable physical
properties, such as enhanced pharmaceutical stability at different
temperatures and humidities, the required solubility in water or
oil, or they are non-toxic.
[0023] Suitable pharmaceutically acceptable salts of substances
according to the invention preferably comprise anions derived from
inorganic acids such as hydrochloric, hydrobromic, hydrofluoric,
boric, fluoboric, phosphoric, metaphosphoric, nitric, carbonic,
sulphurous and sulfuric acids, and organic acids such as acetic
acid, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric,
gluconic, glycolic, isethionic, lactic, lactobionic, maleic,
malonic, methanesulfonic, trifluoromethanesulfonic, succinic,
toluenesulfonic, tartaric, and trifluoroacetic acids. Suitable
organic acids generally include, for example, the following classes
of organic acids: aliphatic, cycloaliphatic, aromatic, araliphatic,
heterocyclic, carboxylic and sulfonic acids.
[0024] Specific examples of suitable organic acids include acetate,
trifluoroacetate, formate, propionate, succinate, glycolate,
gluconate, digluconate, lactate, malate, tartrate, citrate,
ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate,
glutamate, benzoate, anthranilate, stearate, salicylate,
p-hydroxybenzoate, phenylacetate, mandelate, pamoate,
methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate,
toluenesulfonate, 2-hydroxyethanesulfonate, sulfanilate,
cyclohexylaminosulfonate, 13-hydroxybutyrate, galactarate,
galacturonate, adipate, alginate, butyrate, camphorate,
camphorsulfonate, cyclopentanepropionate, dodecylsulfate,
glycoheptanoate, glycerophosphate, heptanoate, hexanoate,
nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate,
3-phenylpropionate, picrate, pivalate, thiocyanate, and
undecanoate.
[0025] Compounds of formula I contain several chiral centers
(particularly on the phosphorus atom and in the R.sub.5 group). The
existence of a chiral center allows the compound to exist as one of
two possible optical isomers ((R)- or (S)-enantiomer) or as a
mixture, typically a racemic mixture, of both. All of the resulting
diastereomers and mixtures of diastereomers are also included
within the scope of lipophosphonoxins of the second generation
general of formula I as described by the invention.
[0026] The invention further includes lipophosphonoxins of general
formula I, or pharmaceutically acceptable salts and/or hydrates
and/or mixtures of such compounds for use as medicaments.
[0027] The invention further includes lipophosphonoxins of general
formula I, or pharmaceutically acceptable salts and/or hydrates
and/or mixtures of such compounds for use as antibacterials.
[0028] The invention further includes an antibacterial drug,
containing lipophosphonoxins of general formula I or their
diastereomers, or pharmaceutically acceptable salts and/or hydrates
and/or mixtures of such compounds as the active ingredient.
[0029] The present invention further includes a method of treatment
of disorders caused by bacteria, comprising the step of
administering at least one lipophosphonoxin of general formula I or
pharmaceutically acceptable salt and/or hydrate thereof to a
subject in need of such treatment.
[0030] Finally, the invention includes the use of lipophosphonoxins
of general formula I or their diastereomers, or pharmaceutically
acceptable salts and/or hydrates and/or mixtures of such compounds
as active ingredients of disinfectants for other than therapeutic
purposes, and/or use as a component of selective culivation media
for in vitro cultures.
[0031] A medicament is any substance or combination of substances
intended for treating or preventing disease in humans or animals
and any substance or combination of substances which may be
administered to humans or animals with a view to making a medical
diagnosis or to restoring, improving or modifying physiological
functions in humans or animals.
[0032] The substances of the invention exhibit antibacterial
activities in particular against strains of Escherichia coli,
Pseudomonas aeruginosa, Enterococcus faecalis, Bacterium subtilis,
and Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus
haemolyticus, Enterococcus faecium, Staphylococcus epidermidis,
Salmonella enteritidis and even against strains resistant to
existing antibiotics.
[0033] Compared with the first generation LPPO (J. Med. Chem. 2011,
54(22), 7884-7898, CZ PV 2011-312, EP2527351), the LPPO of second
generation, which are the object of the present invention, exhibit
a much broader spectrum of antibacterial activity. The greatest
benefit over the prior art is the fact that they are mainly
effective against clinically important gram-negative bacterial
strains such as Escherichia coli, Pseudomonas aeruginosa or
Salmonella enteritidis. Surprisingly, they are also effective
against harmful multiresistant bacterial strains occurring in the
hospital environment, which were not sensitive against the first
generation LPPO.
[0034] The compounds of this invention exhibit little or no effects
on viability of normal human erythroid cells cultured in vitro in
the range of antibacterially active concentrations of the
compounds. The same applies to their induced cytotoxicity.
[0035] Modularity of the structure and easy synthesis by connecting
the individual modules allows large structural variations of the
compounds of this invention, which can lead to modulation of their
biological activity.
EXAMPLES
List of Abbreviations
[0036] DCM dichloromethane [0037] TPSCl
triisopropylbenzenesulfonylchloride [0038] IR infrared spectrum
[0039] HR-ESI high-resolution electrospray ionisation mass spectrum
[0040] HR-EI high-resolution electroimpact ionisation mass spectrum
[0041] n-BuOH n-butylalcohol [0042] DMTr dimethoxytrityl [0043] THF
tetrahydrofuran [0044] EC.sub.50 median active (effective)
concentration (causing 50% of maximum effect) [0045] IC.sub.50
inhibitory concentration (causing 50% of the maximum inhibitory
effect) [0046] rt room temperature [0047] MIC minimum inhibitory
concentration [0048] MBC minimal bactericidal concentration
Example 1
Hexadecyl-uridine-5'-yl-2-N-bis (3-aminopropyl)-2-aminoethyl
phosphonate
##STR00003##
[0050] A mixture of bis boc-N-1-(3-aminopropyl)propane-1,3-diamine
(0.53 g, 1.5 mmol) (prepared according to J. Med. Chem. 2014, 57
(22), 9409-9423) and
hexadecyl-2',3'-isopropylidenuridin-5'-yl-vinylphosphonate (0.6 g,
1 mmol) (prepared according to J. Med. Chem. 2011, 54(22),
7884-7898) in n-BuOH (10 ml) was stirred overnight at 105.degree.
C. The reaction mixture was concentrated in vacuum and the
isopropylidene-protected intermediate was purified by
chromatography on silica gel using a linear gradient of ethanol in
chloroform (0-10%). The resulting solid was dissolved in 0.5
moll.sup.-1 HCl in methanol (40 ml) and the mixture was stirred for
12 hours at room temperature. The reaction mixture was concentrated
to about half volume on rotary evaporator and added to cold ethyl
acetate (20 ml). The solid obtained was filtered and dried. This
resulted in the desired product as an amorphous solid in 74% yield
(0.56 g, 0.74 mmol).
[0051] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.14CH.sub.2O); 1.24-1.43 (m, 52H,
CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 1.71 (m, 4H,
CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 2.15-2.25 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 2.52-2.67 (m, 4H,
PCH.sub.2CH.sub.2N); 3.10 (t, 8H, J.sub.vic=7.5,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.35-3.42 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.44-3.52 (bm, 4H,
PCH.sub.2CH.sub.2N); 4.10-4.21 (m, 8H, H-3',4',
CH.sub.3(CH.sub.2).sub.14CH.sub.2O); 4.27 (dd, 1H, J.sub.2',3'=5.4,
J.sub.2',1'=4.2, H-2'); 4.28 (dd, 1H, J.sub.2',3'=5.3,
J.sub.2',1'=3.9, H-2'); 4.34 (ddd, 1H, J.sub.gem=11.6,
J.sub.H,P=7.5, J.sub.5'b,4'=5.4, H-5'b); 4.39 (dd, 2.sub.H,P=7.6,
J.sub.5',4'=4.3, H-5'); 4.43 (ddd, 1H, J.sub.gem=11.6,
J.sub.H,P=7.3, J.sub.5'a,4'=2.9, H-5'a); 5.77 (d, 2H,
J.sub.5,6=8.0, H-5); 5.84 (d, 1H, J.sub.1',2'=4.2, H-1'); 5.85 (d,
1H, =3.9, H-1'); 7.74 (d, 1H, J.sub.6,5=8.0, H-6); 7.75 (d, 1H,
J.sub.6,5=8.0, H-6).
[0052] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.45
(CH.sub.3(CH.sub.2).sub.14CH.sub.2O); 21.33 (d, J.sub.C,P=140.8,
PCH.sub.2CH.sub.2N); 21.37 (d, J.sub.C,P=141.1,
PCH.sub.2CH.sub.2N); 23.28 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2);
23.73, 26.57, 30.32, 30.47, 30.68, 30.75, 30.76, 30.80
(CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 31.56 (d,
J.sub.C,P=5.9, CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 31.55,
31.56 (d, J.sub.C,P=5.9,
CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 33.07
(CH.sub.3(CH.sub.2).sub.13CH.sub.2CH.sub.2O); 37.87
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 48.58 (NCH.sub.2CH.sub.2P);
51.09 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 67.37 (d, J.sub.C,P=6.1,
CH.sub.2-5'); 68.37 (d, J.sub.C,P=6.8,
CH.sub.3(CH.sub.2).sub.14CH.sub.2O); 68.56 (d, J.sub.C,P=6.8,
CH.sub.3(CH.sub.2).sub.14CH.sub.2O); 70.81, 70.90 (CH-3'); 74.61,
74.65 (CH-2'); 83.37 (d, J.sub.C,P=6.0, CH-4'); 83.39 (d,
J.sub.C,P=6.2, CH-4'); 92.14, 92.26 (CH-1'); 103.17, 103.21 (CH-5);
143.00, 143.04 (CH-6); 152.22, 152.28 (C-2); 165.96, 165.97 (C-4).
.sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.67; 28.13.
[0053] IR v.sub.max(KBr) 3426 (s, vbr), 3047 (m, vbr), 2640 (m,
vbr, sh), 2090 (w, vbr, sh), 1700 (vs, sh), 1681 (vs), 1467 (m),
1429 (w), 1390 (w), 1261 (w, br), 1206 (s), 1080 (w, sh), 1060 (m),
1021 (m, br), 1002 (m), 764 (vw, sh).
[0054] HR-ESI C.sub.33H.sub.65O.sub.8N.sub.5P (M+H).sup.+
calculated 690.45653, found 690.45656.
Example 2
Pentadecyl-uridine-5'-yl-2-N-bis(3-aminopropyl)-2-aminoethyl
phosphonate
##STR00004##
[0056] The compound in Example 2 was prepared by the same procedure
as the one in Example 1 from bis
boc-N-1-(3-aminopropyl)propane-1,3-diamine (0.53 g, 1.5 mmol) and
pentadecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate
(prepared according to J. Med. Chem. 2011, 54(22), 7884-7898) (0.63
g, 1 mmol) in 75% yield (0.56 g, 0.75 mmol).
[0057] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A,B); 1.24-1.43 (m, 48H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A,B); 1.71 (m, 4H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A,B); 2.15-2.23 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 2.52-2.62 (m, 4H,
PCH.sub.2CH.sub.2N-A,B); 3.09 (t, 8H, J.sub.vic=7.5,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.32-3.42 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.43-3.51 (bm, 4H,
PCH.sub.2CH.sub.2N); 4.11-4.20 (m, 8H, H-3',4'-A,B,
CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A,B); 4.26 (dd, 1H, =5.3,
J.sub.2',1'=4.1, H-2'-A); 4.28 (dd, 1H, =5,1, =3.9, H-2'-B);
4.30-4.45 (m, 4H, H-5'-A,B); 5.764 (d, 1H, J.sub.5,6=8.0, H-5-A);
5.766 (d, 1H, J.sub.5,6=8.0, H-5-B); 5.83 (d, 1H, J.sub.1',2'=4.1,
H-1'-A); 5.84 (d, 1H, J.sub.1',2'=3.9, H-1'-B); 7.73 (d, 1H,
J.sub.6,5=8.0, H-6-B); 7.74 (d, 1H, J.sub.6,5=8.0, H-6-A). .sup.13C
NMR (125.7 MHz, CD.sub.3OD): 14.45
(CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A,B); 21.30 (d,
J.sub.C,P=140.7, PCH.sub.2CH.sub.2N-A); 21.35 (d, J.sub.C,P=140.9,
PCH.sub.2CH.sub.2N--B); 23.34
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 23.74, 26.58, 30.33,
30.48, 30.69, 30.75, 30.77, 30.79; 30.81
(CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A,B); 31.56 (d,
J.sub.C,P=5.9, CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A); 31.57
(d, J.sub.C,P=5.9, CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O--B);
33.08 (CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A,B); 37.87
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 48.51
(NCH.sub.2CH.sub.2P-A,B); 51.12
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 67.41 (d, J.sub.C,P=6.3,
CH.sub.2-5'-A); 67.44 (d, J.sub.C,P=6.1, CH.sub.2-5'-B); 68.36 (d,
J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.13CH.sub.2O--B); 68.56 (d,
J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A); 70.84
(CH-3'-B); 70.91 (CH-3'-A); 74.59 (CH-2'-A); 74.63 (CH-2'-B); 83.35
(d, J.sub.C,P=6.1, CH-4'-A,B); 92.31 (CH-1'-A); 92.40 (CH-1'-B);
103.13 (CH-5-A); 103.18 (CH-5-B); 143.03 (CH-6-A); 143.07 (CH-6-B);
152.20 (C-2-A); 152.27 (C-2-B); 165.98 (C-4-A); 165.99 (C-4-B).
[0058] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.65 (A);
28.10 (B).
[0059] IR v.sub.max(KBr) 3050 (s, vbr, sh), 3411 (s, br), 2645 (m,
br), 2924 (vs), 2854 (vs), 2563 (m, br), 2035 (w, br), 1975 (w, br,
sh), 1690 (vs, br), 1624 (m), 1520 (m, br, sh), 1466 (s), 1408 (m),
1386 (m), 1266 (s), 1233 (s, br, sh), 1075 (s, sh), 1055 (s), 1035
(s, br, sh), 997 (s), 822 (m), 764 (w), 721 (w).
[0060] HR-ESI C.sub.32H.sub.63O.sub.8N.sub.5P (M+H).sup.+
calculated 676.44088, found 676.44092.
Example 3
Tetradecyl-uridine-5'-yl-2-N-bis (3-aminopropyl)-2-aminoethyl
phosphonate
##STR00005##
[0062] The compound in Example 3 was prepared by the same procedure
as the one in Example 1 from
boc-N-1-(3-aminopropyl)propane-1,3-diamine (0.62 g, 1.86 mmol) and
tetradecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate
(prepared according to J. Med. Chem. 2011, 54(22), 7884-7898) (0.76
g, 1.33 mmol) in 65% yield (0.64 g, 0.87 mmol).
[0063] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 1.24-1.43 (m, 44H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 1.71 (m, 4H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O-A,B); 2.16-2.26 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 2.54-2.65 (m, 4H,
PCH.sub.2CH.sub.2N-A,B); 3.10 (t, 8H, J J.sub.vic=7.5,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.35-3.42 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.44-3.52 (bm, 4H,
PCH.sub.2CH.sub.2N); 4.08-4.22 (m, 8H, H-3',4'-A,B,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 4.27 (dd, 1H,
J.sub.2',3'=5.4, J.sub.2',1'=4.2, H-2'-B); 4.28 (dd, 1H,
J.sub.2',3'=5.3, J.sub.2',1'=3.9, H-2'-A); 4.34 (ddd, 1H,
J.sub.gem=11.5, J.sub.H,P=7.4, J.sub.5'b,4'=5.4, H-5'b-B); 4.39
(dd, 2H, J.sub.H,P=7.6, J.sub.5',4'=4.2, H-5'-A); 4.43 (ddd, 1H,
J.sub.gem=11.6, J.sub.H,P=7.2, J.sub.5'a,4'=2.9, H-5'a-B); 5.78 (d,
2H, J.sub.5,6=8.0, H-5-A,B); 5.84 (d, 1H, J.sub.1',2'=4.2, H-1'-B);
5.85 (d, 1H, J.sub.1',2'=3.9, H-1'-A); 7.74 (d, 1H, J.sub.6,5=8.0,
H-6-A); 7.75 (d, 1H, J.sub.6,5=8.0, H-6-B).
[0064] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.45
(CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 21.33 (d,
J.sub.C,P=140.7, PCH.sub.2CH.sub.2N--B); 21.38 (d, J.sub.C,P=141.1,
PCH.sub.2CH.sub.2N-A); 23.28
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 23.73, 26.57, 30.32,
30.48, 30.68, 30.74, 30.76, 30.78, 30.79, 30.81
(CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 31.55 (d,
J.sub.C,P=5.8, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O--B);
31.56 (d, J.sub.C,P=5.9,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A); 33.07
(CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 37.87
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 48.59
(NCH.sub.2CH.sub.2P-A,B); 51.09
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 67.36 (d, J.sub.C,P=6.2,
CH.sub.2-5'-A); 67.37 (d, J.sub.C,P=6.2, CH.sub.2-5'-B); 68.37 (d,
J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.12CH.sub.2O--B); 68.56 (d,
J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A); 70.81
(CH-3'-A); 70.90 (CH-3'-B); 74.61 (CH-2'-B); 74.66 (CH-2'-A); 83.37
(d, J.sub.C,P=5,9, CH-4'-A); 83.39 (d, J.sub.C,P=6.1, CH-4'-B);
92.12 (CH-1'-B); 92.24 (CH-1'-A); 103.17 (CH-5-B); 103.21 (CH-5-A);
142.99 (CH-6-B); 143.04 (CH-6-A); 152.23 (C-2-B); 152.28 (C-2-A);
165.96 (C-4-B); 165.97 (C-4-A).
[0065] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.66 (B);
28.12 (A).
[0066] IR v.sub.max(CHCl.sub.3) 3415 (s, vbr), 3045 (s, vbr, sh),
2924 (vs), 2854 (s), 2644 (m, vbr), 2563 (m, vbr), 2028 (w, vbr),
1972 (w, vbr, sh), 1690 (vs, br), 1624 (m), 1520 (w, br, sh), 1465
(s), 1407 (m), 1386 (m), 1266 (s), 1232 (s, sh), 1072 (s, sh), 1054
(s), 1015 (s, sh), 996 (s), 823 (m), 763 (w), 721 (w).
[0067] HR-ESI C.sub.31H.sub.61O.sub.8N.sub.5P (M+H).sup.+
calculated 662.42523, found 662.42502.
Example 4
Tridecyl-uridine-5'-yl-2-N-bis (3-aminopropyl)-2-aminoethyl
phosphonate
##STR00006##
[0069] The compound in Example 4 was prepared by the same procedure
as the one in Example 1 from
boc-N-1-(3-aminopropyl)propane-1,3-diamine (0.95 g, 2.85 mmol) and
tridecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate
(prepared according to J. Med. Chem. 2011, 54(22), 7884-7898) (1.32
g, 2.38 mmol) in 65% yield (1.11 g, 1.54 mmol).
[0070] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2O); 1.24-1.43 (m, 40H,
CH.sub.3(CH.sub.2).sub.10CH.sub.2CH.sub.2O); 1.68-1.75 (m, 4H,
CH.sub.3(CH.sub.2).sub.10CH.sub.2CH.sub.2O); 2.18 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 2.55 (m, 4H,
PCH.sub.2CH.sub.2N); 3.09 (t, 8H, J.sub.vic=7.4,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.35 (m, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.45 (m, 4H,
PCH.sub.2CH.sub.2N); 4.09-4.20 (m, 8H, H-3',4',
CH.sub.3(CH.sub.2).sub.11CH.sub.2O); 4.26 (dd, 1H, J.sub.2'3'=5.2,
J.sub.2',1'=4.0, H-2'); 4.28 (dd, 1H, J.sub.2',3'=5.1,
J.sub.2',1'=3.9, H-2'); 4.33 (ddd, 1H, J.sub.gem=11.4,
J.sub.H,P=7.6, J.sub.5'b,4'=5.3, H-5'b); 4.38 (dd, 2H,
J.sub.H,P=7.6, J.sub.5',4'=4.2, H-5'); 4.43 (ddd, 1H, J=11.4,
J.sub.H,P=7.4, J.sub.5'a,4'=2.9, H-5'a); 5.759, 5.761 (2.times.d,
2.times.1H, J.sub.5,6=8.1, H-5); 5.83 (d, 1H, J.sub.1',2'=4.0,
H-1'); 5.84 (d, 1H, J.sub.1',2'=3.9, H-1'); 7.72, 7.73 (2.times.d,
2.times.1H, J.sub.6,5=8.1, H-6).
[0071] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.44
(CH.sub.3(CH.sub.2).sub.11CH.sub.2O); 20.34 (d, J.sub.C,P=141.8,
PCH.sub.2CH.sub.2N); 23.40 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2);
23.74; 26.58; 30.32; 30.48; 30.68; 30.74; 30.77, 30.78, 30.80
(CH.sub.3(CH.sub.2).sub.10CH.sub.2CH.sub.2O); 31.57, 31.58 (d,
J.sub.C,P=6.0, CH.sub.3(CH.sub.2).sub.10CH.sub.2CH.sub.2O); 33.08
(CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2N); 37.91
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 48.66 (PCH.sub.2CH.sub.2N);
51.17 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 67.41, 67.47 (d,
J.sub.C,P=6.3, CH.sub.2-5'); 68.34, 68.55 (d, J.sub.C,P=6.9,
CH.sub.3(CH.sub.2).sub.10CH.sub.2CH.sub.2O); 70.87; 70.93 (CH-3');
74.59; 74.63 (CH-2'); 83.35, 83.36 (d, J.sub.C,P=6.2, CH-4');
92.40, 92.47 (CH-1'); 103.13, 103.19 (CH-5); 143.03, 143.07 (CH-6);
152.20, 152.28 (C-2); 165.95, 165.96 (C-4).
[0072] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.57,
28.01.
[0073] IR v.sub.max(KBr) 3424 (s, br), 3047 (br, sh), 2925 (vs),
2854 (s), 2642 (m, br), 2562 (w, br), 2030 (vw, vbr), 1975 (vw,
vbr), 1690 (vs), 1465 (m), 1406 (m), 1385 (m), 1266 (m), 1232 (m,
sh), 1075 (m, sh), 1054 (m, br), 1035 (m, vbr), 996 (m), 821 (w),
764 (w), 721 (w). HR-ESI C.sub.30H.sub.59O.sub.8N.sub.5P
(M+H).sup.+ calculated 648.409583, found 648.409712.
Example 5
Dodecyl-uridine-5'-yl-2-N-bis (3-aminopropyl)-2-aminoethyl
phosphonate
##STR00007##
[0075] The compound in Example 5 was prepared by the same procedure
as the one in Example 1 from
boc-N-1-(3-aminopropyl)propane-1,3-diamine (0.53 g, 1.5 mmol) and
dodecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate (prepared
according to J. Med. Chem. 2011, 54(22), 7884-7898) (0.55 g, 1
mmol) in 41% yield (0.29 g, 0.41 mmol).
[0076] .sup.1H NMR (600.1 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.10CH.sub.2O); 1.25-1.42 (m, 36H,
CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2O); 1.71 (m, 4H,
CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2O); 2.22 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 2.60 (m, 4H,
PCH.sub.2CH.sub.2N); 3.11 (t, 8H, J.sub.vic=7.4,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.39 (m, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 3.48 (m, 4H,
PCH.sub.2CH.sub.2N); 4.08-4.22 (m, 8H, H-3',4',
CH.sub.3(CH.sub.2).sub.10CH.sub.2O); 4.27 (dd, 1H, J.sub.2',3'=5.5,
=4.2, H-2'); 4.29 (dd, 1H, J.sub.2',3'=5.4, =4.0, H-2'); 4.34 (ddd,
1H, J.sub.gem=11.4, J.sub.H,P=7.5, J.sub.5'b,4'=5.4, H-5'b); 4.39
(dd, 2H, J.sub.H,P=7.5, J.sub.5'4'=4.3, H-5'); 4.44 (ddd, 1H,
J=11.4, J.sub.H,P=7.3, J.sub.5'a,4'=2.9, H-5'a); 5.78 (d, 2H,
J.sub.5,6=8.1, H-5); 5.84 (d, 1H, J.sub.1',2'=4.2, H-1'); 5.85 (d,
1H, J.sub.1',2'=4,0, H-1'); 7.73, 7.74 (2.times.d, 2.times.1H,
J.sub.6,5=8.1, H-6).
[0077] .sup.13C NMR (150.9 MHz, CD.sub.3OD): 14.43
(CH.sub.3(CH.sub.2).sub.10CH.sub.2O); 20.38, 21.42 (d,
J.sub.C,P=140.9, PCH.sub.2CH.sub.2N); 23.28
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 23.71; 26.56; 30.30; 30.46;
30.66; 30.72; 30.74, 30.76
(CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2O); 31.55, 31.56 (d,
J.sub.C,P=5.9, CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2O); 33.05
(CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2N); 37.90
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 48.65 (PCH.sub.2CH.sub.2N);
51.13 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2); 67.35, 67.37 (d,
J.sub.C,P=6.2, CH.sub.2-5'); 68.39, 68.57 (d, J.sub.C,P=6.8,
CH.sub.3(CH.sub.2).sub.9CH.sub.2CH.sub.2O); 70.83; 70.92 (CH-3');
74.61; 74.66 (CH-2'); 83.40, 83.42 (d, J.sub.C,P=6.1, CH-4') 92.13,
92.25 (CH-1'); 103.19, 103.23 (CH-5); 142.98, 143.02 (CH-6);
152.23, 152,28 (C-2); 165.93, 165.94 (C-4).
[0078] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.60,
28.05.
[0079] IR v.sub.max(KBr) 3391 (s, br), 3000 (vs, vbr), 2925 (vs),
2854 (vs), 2645 (s, br), 2563 (m, br), 2031 (w, br), 1692 (vs, br),
1623 (m), 1575 (w, sh), 1515 (m, br, sh), 1465 (s), 1408 (m), 1385
(m), 1267 (s), 1233 (s, sh), 1077 (s, br, sh), 1058 (s, br, sh),
1036 (s, br), 998 (s, br), 823 (m), 763 (w), 721 (w).
[0080] HR-ESI C.sub.29H.sub.57O.sub.8N.sub.5P (M+H).sup.+
calculated 634.39393, found 634.39398.
Example 6
Pentadecyl-uridine-5'-yl-2-N-bis(3-aminoethyl)-2-aminoethyl
phosphonate
##STR00008##
[0082] The compound in Example 6 was prepared by the same procedure
as the one in Example 1 from
boc-N-1-(2-aminoetyl)ethane-1,2-diamine (0.2 g, 0.66 mmol)
(prepared according to J. Med. Chem. 2014, 57 (22), 9409-9423) and
pentadecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate
(prepared according to J. Med. Chem. 2011, 54(22), 7884-7898) (0.29
g, 0.5 mmol) in 50% yield (0.19 g, 0.25 mmol).
[0083] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.13CH.sub.2O); 1.23-1.44 (m, 48H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 1.70 (m, 4H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 2.16-2.29 (m, 4H,
PCH.sub.2CH.sub.2N); 2.83-3.00 (m, 12H, NCH.sub.2CH.sub.2NH.sub.2,
PCH.sub.2CH.sub.2N); 3.09-3.17 (bm, 8H, NCH.sub.2CH.sub.2NH.sub.2);
4.06-4.20 (m, 8H, H-3',4', CH.sub.3(CH.sub.2).sub.13CH.sub.2O);
4.24-4.43 (m, 6H, H-2',5'); 5.755 (d, 1H, J.sub.5,6=8.0, H-5);
5.757 (d, 1H, J.sub.5,6=8.0, H-5); 5.808 (d, 1H, J.sub.1',2'=3.7,
H-1'); 5.812 (d, 1H, J.sub.1',2'=3.9, H-1'); 7.71 (d, 1H,
J.sub.6,5=8.0, H-6); 7.72 (d, 1H, J.sub.6,5=8.0, H-6).
[0084] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.45
(CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A,B); 22.78 (d,
J.sub.C,P=138.3, PCH.sub.2CH.sub.2N); 22.84 (d, J.sub.C,P=138.2,
PCH.sub.2CH.sub.2N); 23.74, 26.64, 30.34, 30.48, 30.70, 30.75,
30.77, 30.79; 30.81 (CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O);
31.57 (d, J.sub.C,P=6.0,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 31.59 (d,
J.sub.C,P=6.0, CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 33.08
(CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 37.90, 37.95
(NCH.sub.2CH.sub.2NH.sub.2); 47.34 (NCH.sub.2CH.sub.2P); 51.47,
51.53 (NCH.sub.2CH.sub.2NH.sub.2); 66.78 (d, J.sub.C,P=6.3,
CH.sub.2-5'); 66.95 (d, J.sub.C,P=6.6, CH.sub.2-5'); 67.89 (d,
J.sub.C,P=6.9, CH.sub.3(CH.sub.2).sub.13CH.sub.2O); 67.93 (d,
J.sub.C,P=6.9, CH.sub.3(CH.sub.2).sub.13CH.sub.2O); 70.81, 70.86
(CH-3'); 74.54, 74.63 (CH-2'); 83.34 (d, J.sub.C,P=6.2, CH-4');
83.39 (d, J.sub.C,P=6.2, CH-4'); 92.66, 92.68 (CH-1'); 103.04,
103.05 (CH-5); 143.05, 143.09 (CH-6); 152.17, 152.20 (C-2); 165.99,
165.99 (C-4).
[0085] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 33.66.
[0086] v.sub.max(KBr) 3423 (s, vbr), 3018 (s, vbr, sh), 2924 (vs),
2854 (vs), 2650 (m, vbr, sh), 2560 (m, vbr), 2032 (vw, vbr), 1946
(vw, vbr), 1691 (s, br), 1626 (m), 1466 (s), 1406 (m), 1387 (m),
1266 (m), 1237 (m, br, sh), 1074 (m, sh), 1052 (m, sh), 1021 (s,
br), 1000 (m, br, sh), 822 (w), 767 (w), 722 (w).
[0087] HR-ESI C.sub.30H.sub.59O.sub.8N.sub.5P (M+H).sup.+
calculated 648.40958, found 648.40969.
Example 7
Tetradecyl-uridine-5'-yl-2-N-bis(3-aminoethyl)-2-aminoethyl
phosphonate
##STR00009##
[0089] The compound in Example 7 was prepared by the same procedure
as the one in Example 1 from
boc-N-1-(2-aminoetyetyl)ethane-1,2-diamine (0.4 g, 1.32 mmol) and
tetradecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate (0.63
g, 1.1 mmol) (prepared according to J. Med. Chem. 2011, 54(22),
7884-7898) in 37% yield (0.27 g, 0.41 mmol).
[0090] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 1.13-1.46 (m, 44H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 1.71 (m, 4H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 2.28-2.49 (m, 4H,
PCH.sub.2CH.sub.2N-A,B); 3.03-3.23 (m, 12H,
NCH.sub.2CH.sub.2NH.sub.2-A,B, PCH.sub.2CH.sub.2N-A,B); 3.23-3.33
(bm, 8H, NCH.sub.2CH.sub.2NH.sub.2-A,B); 4.06-4.22 (m, 8H,
H-3',4'-A,B, CH.sub.3(CH.sub.2).sub.13CH.sub.2O-A,B); 4.24-4.44 (m,
6H, H-2',5'-A,B); 5.76 (d, 2H, J.sub.5,6=8.0, H-5-A,B); 5.83 (d,
2H, J.sub.1',2'=4.0, H-1'-A,B); 7.73 (d, 1H, J.sub.6,5=8.0, H-6-B);
7.74 (d, 1H, J.sub.6,5=8.0, H-6-A).
[0091] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.45
(CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 22.35 (d,
J.sub.C,P=140.4, PCH.sub.2CH.sub.2N--B); 22.39 (d, J.sub.C,P=141.7,
PCH.sub.2CH.sub.2N-A); 23.74, 26.62, 30.34, 30.49, 30.69, 30.75,
30.77, 30.79, 30.80, 30.81
(CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 31.56 (d,
J.sub.C,P=6.0, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A); 31.58
(d, J.sub.C,P=6.0, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O--B);
33.08 (CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 37.07
(NCH.sub.2CH.sub.2NH.sub.2-A,B); 48.03 (NCH.sub.2CH.sub.2P); 51.48
(NCH.sub.2CH.sub.2NH.sub.2--B); 51.51
(NCH.sub.2CH.sub.2NH.sub.2-A); 66.95 (d, J.sub.C,P=6.5,
CH.sub.2-5'-B); 67.05 (d, J.sub.C,P=6.3, CH.sub.2-5'-A); 68.08 (d,
J.sub.C,P=7.3, CH.sub.3(CH.sub.2).sub.12CH.sub.2O)--B); 68.15 (d,
J.sub.C,P=7.4, CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A); 70.78
(CH-3'-B); 70.86 (CH-3'-A); 74.55 (CH-2'-A); 74.64 (CH-2'-B); 83.36
(d, J.sub.C,P=6.0, CH-4'-B); 83.41 (d, J.sub.C,P=6.2, CH-4'-A);
92.46 (CH-1'-A); 92.51 (CH-1'-B); 103.11 (CH-5-A,B); 143.07
(CH-6-B); 143.09 (CH-6-A); 152.21 (C-2-A); 152.25 (C-2-B); 165.95
(C-4-A); 165.98 (C-4-B).
[0092] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 31.69
(A,B).
[0093] IR v.sub.max(KBr) 3427 (s, br), 3000 (s, vbr), 2956 (s),
2924 (vs), 2854 (s), 2560 (m, vbr), 2040 (vw, vbr), 1691 (s), 1466
(m), 1407 (w), 1387 (w), 1267 (m), 1235 (m, br, sh), 1073 (m, sh),
1051 (m, sh), 1018 (m), 1003 (m, sh), 824 (w), 766 (w, sh), 721
(vw).
[0094] HR-ESI C.sub.29H.sub.57O.sub.8N.sub.5P (M+H).sup.+
calculated 634.39393, found 634.39391.
Example 8
Pentadecyl-uridine-5'-yl-2-N-bis(3-guanidinoethyl)-2-aminoethyl
phosphonate
##STR00010##
[0096] A mixture of 1H-pyrazol-1-carboxamidinuhydrochloride (0.24
g, 1.67 mol), the compound from Example 2 (0.5 g, 0.67 mmol) and
dietylisopropylamine (0.57 mlL, 3.35 mmol) in DMF (10 ml) was
stirred under argon at rt overnight. The solvent was evaporated and
the product was obtained after reverse phase chromatography using a
linear gradient of methanol in water (10-100%), evaporation and
reprecipitation with ethyl acetate (50 ml) from a solution in 0.5
moll.sup.-1 HCl in methanol (20 ml) in 64% yield (0.36 g, 0.43
mmol) as an amorphous solid.
[0097] .sup.1H NMR (600.1 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.13CH.sub.2O); 1.25-1.43 (m, 48H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 1.68-1.75 (m, 4H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 2.04-2.13 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH); 2.48-2.60 (m, 4H,
PCH.sub.2CH.sub.2N); 3.26-3.37 (m, 16H,
NCH.sub.2CH.sub.2CH.sub.2NH); 3.40-3.49 (m, 4H,
PCH.sub.2CH.sub.2N); 4.10-4.20 (m, 8H, H-3',4',
CH.sub.3(CH.sub.2).sub.12CH.sub.2O); 4.25-4.29 (m, 2H, H-2'); 4.33
(ddd, 1H, J.sub.gem=11.4, J.sub.H,P=7.5, J.sub.5'b,4'=5.3, H-5'b);
4.38 (dd, 2H, J.sub.H,P=7.5, J.sub.5',4'=4.2, H-5'); 4.43 (ddd, 1H,
J=11.4, J.sub.H,P=7.1, J.sub.5'a,4'=2.9, H-5'a); 5.760, 5.763
(2.times.d, 2.times.1H, J.sub.5,6=8.0, H-5); 5.83 (d, 1H,
J.sub.1',2'=4.2, H-1'); 5.84 (d, 1H, J.sub.1',2'=3.9, H-1'); 7.724,
7.727 (2.times.d, 2.times.1H, J.sub.6,5=8.0, H-6).
[0098] .sup.13C NMR (150.9 MHz, CD.sub.3OD): 14.43
(CH.sub.3(CH.sub.2).sub.13CH.sub.2O); 21.36 (d, J.sub.C,P=139.6,
PCH.sub.2CH.sub.2N); 23.73 (NCH.sub.2CH.sub.2CH.sub.2NH.sub.2);
24.82, 26.59, 30.30, 30.31, 30.47, 30.68, 30.74, 30.76, 30.78,
30.79 (CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 31.56 (d,
J.sub.C,P=5.9, CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 33.07
(CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2N); 39.70
(NCH.sub.2CH.sub.2CH.sub.2NH); 48.40 (d, J.sub.C,P=5.4,
PCH.sub.2CH.sub.2N); 51.66 (NCH.sub.2CH.sub.2CH.sub.2NH); 67.35 (d,
J.sub.C,P=6.4, CH.sub.2-5'); 67.43 (d, J.sub.C,P=6.3, CH.sub.2-5');
68.33 (d, J.sub.C,P=6.9,
CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 68.53 (d,
J.sub.C,P=6.7, CH.sub.3(CH.sub.2).sub.12CH.sub.2CH.sub.2O); 70.84,
70.88 (CH-3'); 74.62, 74.68 (CH-2'); 83.38, 83.43 (2.times.d,
J.sub.C,P=6.1, CH-4'); 92.37, 92.41 (CH-1'); 103.14, 103.19 (CH-5);
143.02, 143.03 (CH-6); 152.19, 152.25 (C-2); 158.69 (C-guanidine);
165.964, 165.970 (C-4).
[0099] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.79,
28.23.
[0100] IR v.sub.max(KBr) 3424 (vs, vbr), 3260 (s, br, sh), 3183 (s,
br), 2700 (w, vbr), 1694 (s, sh), 1671 (s), 1646 (s, sh), 1624 (s,
sh), 1466 (m), 1387 (w), 1268 (m), 1223 (m, br), 1076 (w, sh), 1056
(m), 1036 (m, vbr), 1000 (m), 762 (w, br), 721 (w).
[0101] HR-ESI C.sub.34H.sub.67O.sub.8N.sub.9P (M+H).sup.+
calculated 760.48447, found 760.48452.
Example 9
Tetradecyl-uridine-5'-yl-2-N-bis(3-guanidinoethyl)-2-aminoethyl
phosphonate
##STR00011##
[0103] The compound in Example 9 was prepared by the same procedure
as the compound in Example 8 from
1H-pyrazole-1-carboxamidinehydrochloride (1.8 g, 12.25 mmol), the
compound from Example 3 (3 g, 4.1 mmol) and diethylisopropylamine
(4, 2 ml, 24.6 mmol) in DMF (40 ml) in 59% yield (2.06 g, 2.41
mmol) as an amorphous solid.
[0104] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 1.25-1.43 (m, 44H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 1.68-1.75 (m, 4H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 2.04-2.13 (bm, 8H,
NCH.sub.2CH.sub.2CH.sub.2NH-A,B); 2.52-2.64 (m, 4H,
PCH.sub.2CH.sub.2N-A,B); 3.32-3.37 (m, 16H,
NCH.sub.2CH.sub.2CH.sub.2NH-A,B); 3.42-3.50 (m, 4H,
PCH.sub.2CH.sub.2N-A,B); 4.11-4.20 (m, 8H, H-3',4'-A,B,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 4.26 (dd, 1H,
J.sub.2',3'=5.3, J.sub.2',1'=4.2, H-2'-B); 4.27 (dd, 1H,
J.sub.2',3'=5,2, J.sub.2',1'=3.9, H-2'-A); 4.33 (ddd, 1H,
J.sub.gem=11.4, J.sub.H,P=7.5, J.sub.5b',4'=5.3, H-5'b-B); 4.38
(dd, 2H, J.sub.H,P=7.5, J.sub.5',4'=4.2, H-5'-A); 4.43 (ddd, 1H,
J=11.4, J.sub.H,P=7.1, J.sub.5'a,4'=2.9, H-5'a-B); 5.77 (d, 2H,
J.sub.5,6=8.1, H-5-A,B); 5.84 (d, 1H, J.sub.1',2'=4.2, H-1'-B);
5.85 (d, 1H, J.sub.1',2'=3.9, H-1'-A); 7.747 (d, 1H, J.sub.6,5=8.1,
H-6-A); 7.752 (d, 1H, J.sub.6,5=8.1, H-6-B).
[0105] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.47
(CH.sub.3(CH.sub.2).sub.12CH.sub.2O); 21.32 (d, J.sub.C,P=141.3,
PCH.sub.2CH.sub.2N-A,B); 23.76
(NCH.sub.2CH.sub.2CH.sub.2NH.sub.2-A,B); 24.76, 26.60, 30.33,
30.51, 30.71, 30.77, 30.79, 30.81, 30.83, 30.84
(CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 31.56 (d,
J.sub.C,P=6,1, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B);
33.09 (CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2N-A,B); 39.64
(NCH.sub.2CH.sub.2CH.sub.2NH-A,B); 48.38 (PCH.sub.2CH.sub.2N-A,B);
51.56 (NCH.sub.2CH.sub.2CH.sub.2NH-A,B); 67.31 (d, J.sub.C,P=6.5,
CH.sub.2-5'-B); 67.37 (d, J.sub.C,P=6.5, CH.sub.2-5'-A); 68.31 (d,
J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A); 68.51
(d, J.sub.C,P=6.8, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O--B);
70.78 (CH-3'-A); 70.84 (CH-3'-B); 74.63 (CH-2'-B); 74.70 (CH-2'-A);
83.37 (d, J.sub.C,P=5.6, CH-4'-B); 83.42 (d, J.sub.C,P=5.6,
CH-4'-B); 92.13 (CH-1'-B); 92.20 (CH-1'-A); 103.13 (CH-5-B); 103.17
(CH-5-A); 143.00 (CH-6-B); 143.03 (CH-6-A); 152.19 (C-2-B); 152.24
(C-2-A); 158.62 (C-guanidine-A,B); 165.99 (C-4-B); 166.00
(C-4-A),
[0106] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 27.83 (P-B);
28.28 (P-A).
[0107] IR v.sub.max(KBr) 3320 (s, vbr), 3260 (s, vbr), 3155 (s,
vbr), 2925 (s), 2854 (s), 2710 (m, vbr), 2604 (m), 2502 (m, vbr,
sh), 1669 (vs, vbr), 1622 (vs, sh), 1465 (s), 1407 (m), 1379 (s),
1265 (s), 1235 (s, br, sh), 1075 (s, br, sh), 1045 (s), 1016 (s,
br), 1002 (s, sh), 822 (m), 720 (w), 580 (m, vbr), 490 (m, br,
sh).
[0108] HR-ESI C.sub.33H.sub.65O.sub.8N.sub.9P (M+H).sup.+
calculated 746.46882, found 746.46902.
Example 10
Tetradecyl-uridine-5'-yl-(3-aminopyrrolidin-1-N-yl) ethyl
phosphonate
##STR00012##
[0110] The compound in Example 10 was prepared by the same
procedure as the one in Example 1 from 3-boc-3-aminopyrrolidine
(0.51 g, 2.75 mmol) and
tetradecyl-2',3'-isopropylidenuridine-5'-yl-vinylphosphonate (1.31
g, 2.3 mmol) (prepared according to J. Med. Chem. 2011, 54(22),
7884-7898) in 26% yield (0.384 g, 0.59 mmol).
[0111] .sup.1H NMR (500.0 MHz, CD.sub.3OD): 0.90 (m, 6H,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 1.24-1.43 (m, 44H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 1.65-1.73 (m, 4H,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 1.94-2.02 (bm, 2H,
H-4b-pyrrolidine-A,B); 2.12-2.25 (bm, 6H, H-4b-pyrrolidine-A,B,
PCH.sub.2CH.sub.2N-A,B); 2.89-3.02 (bm, 4H,
PCH.sub.2CH.sub.2N-A,B); 3.24 (bdd, 2H, J.sub.gem=12.2,
J.sub.2b,3=3.5, H-2b-pyrrolidine-A,B); 3.29-3.36 (m, 4H,
H-2a,5b-pyrrolidine-A,B); 3.43-3.49 (m, 2H, H-5a-pyrrolidine-A,B);
3.62 (bm, 2H, H-3-pyrrolidine-A,B); 4.04-4.17 (m, 8H, H-3',4'-A,B,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 4.21-4.39 (m, 6H,
H-2',5'-A,B); 5.73 (d, 2H, J.sub.5,6=8.0, H-5-A,B); 5.83 (d, 2H,
J.sub.1',2'=3.9, H-1'-A,B); 7.71 (d, 1H, J.sub.6,5=8.0, H-6-B);
7.72 (d, 1H, J.sub.6,5=8.0, H-6-A).
[0112] .sup.13C NMR (125.7 MHz, CD.sub.3OD): 14.44
(CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A,B); 23.74
(CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 26.08 (d,
J.sub.C,P=139.9, PCH.sub.2CH.sub.2N-A,B); 26.65, 26.66, 30.29,
30.30, 30,48, 30.67, 30.68, 30,72, 30,73, 30,77, 30.78, 30.79,
30.81 (CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 31.03
(CH.sub.2-4-pyrrolidine-A,B); 31.57 (b, J.sub.C,P=6.1,
CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A); 31.58 (d,
J.sub.C,P=6.1, CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O--B);
33.08 (CH.sub.3(CH.sub.2).sub.11CH.sub.2CH.sub.2O-A,B); 42.13 (d,
J.sub.C,P=2.5, NCH.sub.2CH.sub.2P-A,B); 45.29
(CH.sub.2-5-pyrrolidine-A,B); 50.72, 50.79
(CH.sub.2-2-pyrrolidine-A,B); 57.60 (CH-3-pyrrolidine-B); 57.63
(CH-3-pyrrolidine-A); 66.55 (d, J.sub.C,P=6.6, CH.sub.2-5'-A);
66.63 (d, J.sub.C,P=6.5, CH.sub.2-5'-B); 67.68 (d, J.sub.C,P=6.9,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O--B); 67.78 (d, J.sub.C,P=6.9,
CH.sub.3(CH.sub.2).sub.12CH.sub.2O-A); 70.84 (CH-3'-A,B); 74.83
(CH-2'-A); 74.86 (CH-2'-B); 83.48 (d, J.sub.C,P=6.4, CH-4'-A);
83.51 (d, J.sub.C,P=6.3, CH-4'-B); 92.16 (CH-1'-A); 92.24
(CH-1'-B); 102.97 (CH-5-A,B); 142.75 (CH-6-A); 142.78 (CH-6-B);
152.16 (C-2-A); 152.17 (C-2-B); 165.98 (C-4-A); 165.99 (C-4-B).
[0113] .sup.31P{.sup.1H} NMR (202.3 MHz, CD.sub.3OD): 32.16 (A);
32.36 (B).
[0114] IR v.sub.max(CHCl.sub.3) 3415 (s, vbr), 3051 (s, br), 2924
(vs), 2854 (vs), 2755 (m, vbr, sh), 2455 (w, vbr), 2030 (vw, vbr),
1970 (vw, vbr), 1693 (vs, br), 1464 (s), 1405 (m, sh), 1385 (m),
1261 (s, br), 1224 (m), 1075 (s, sh), 1053 (s), 1036 (s, sh), 1019
(s, sh), 996 (s), 822 (m), 766 (w), 721 (w).
[0115] HR-ESI C.sub.29H.sub.54O.sub.8N.sub.4P (M+H).sup.+
calculated 617.36738, found 617.36742.
[0116] Antibacterial Activity
[0117] Antibacterial activity was measured using a standard
microdilution method, showing the minimum inhibitory concentration
(MIC) of the test sample which results in inhibition of bacterial
growth. Disposable microtiter plates were used for the tests.
Samples are dissolved in the brain-heart infusion broth (HiMedia
Laboraties Pvt. Ltd., Czech Republic), and Mueller Hinton broth
(HiMedia Laboraties, see above) at a final concentration ranging
from 200 .mu.g/ml to 1.5625 .mu.g/ml. Plates were inoculated with a
standard amount of test bacteria--inoculum density in the hole
corresponds to 10.sup.5-6 CFU/ml (colony forming units/ml). MIC
values are read after 24/48 hours of incubation at 37.degree. C. as
the minimum inhibitory concentration of the test substance at which
the growth of bacteria is inhibited. Minimal bactericidal
concentration (MBC) is defined as the minimum concentration of the
sample needed to achieve irreversible inhibition, therefore killing
the bacteria after a defined time of incubation. The MBC was
determined by inoculation method. 10 .mu.l from the wells in a
microplate with a defined concentration of test substance is taken
with an applicator, and inoculated onto the surface of blood agar
(Trios, Czech Republic) and Sabouraud agar (Trios, CR). The MBC was
determined as the lowest concentration that inhibited visible
growth of the bacteria used.
[0118] Standard reference bacterial strains (Escherichia coli CCM
3954, Pseudomonas aeruginosa CCM 3955, Enterococcus faecalis CCM
4224, Staphylococcus aureus CCM 4223) were obtained from the Czech
Collection of Microorganisms (CCM) at Masaryk University in Brno.
Streptococcus agalactiae, Bacillus subtilis were obtained from the
University Hospital Olomouc. The tested microorganisms were
maintained in cryobanks (ITEST plus, Czech Republic) at -80.degree.
C.
TABLE-US-00001 TABLE 1 Minimum inhibitory concentrations of
lipophosphonoxins of the present invention against a panel of
reference bacterial strains MIC .mu.g/ml Pseudomonas Enterococcus
Staphylococcus Compound Escherichia coli aeruginosa faecalis aureus
Streptococcus from example CCM 3954 CCM 3955 CCM 4224 CCM 4223
Bacillus subtilis agalactiae 1 3.125 6.25 50 12.5 0.78 3.125 2 6.25
3.125 50 6.25 1.56 3.125 3 6.25 0.78 25 6.25 0.78 3.125 4 25 3.125
50 12.5 3.125 6.25 5 25 3.125 100 25 3.125 12.5 6 1.56 1.56 12.5
6.25 0.78 3.125 7 12.5 3.125 100 25 6.25 6.25 8 0.78 0.78 25 3.125
0.39 1.56 9 3.125 3.125 12.5 6.25 1.56 3.125 10 3.125 3.125 6.25
12.5 1.56 3.125
TABLE-US-00002 TABLE 2 Minimum inhibitory concentrations of
lipophosphonoxins of the present invention against a panel of
reference bacterial strains MIC .mu.g/ml Burkholderia Compound
Salmonella Acinetobacter Stenotrophomonas multivorans ATCC from
example Enteritidis S2-25 baumanii A3-08 matophilia S2-23 BAA-247 2
3.125 6.25 3.125 12.5 3 6.25 25 50 200 4 50 50 50 50 7 12.5 25 6.25
12.5 8 1.56 6.25 3.125 100 9 3.125 12.5 12.5 100
TABLE-US-00003 TABLE 3 Minimum inhibitory concentrations of some of
lipophosphonoxins of the present invention against a panel of
resistant bacterial strains MIC .mu.g/ml Compound E. coli P.
aeruginosa S. aureus S. haemolyticus E. faecium S. epidermidis from
example 16702 16575 MRSA 4591 16568 VanA 419ana 8700B 1 3.125 3.125
25 3.125 25 3.125 2 6.25 3.125 12.5 3.125 25 6.25 3 3.125 1.56 6.25
1.56 100 1.56 4 50 25 25 6.25 100 12.5 5 25 12.5 50 12.5 200 12.5 6
1.56 1.56 6.25 1.56 100 1.56 7 6.25 6.25 12.5 3.125 100 3.125 8
0.78 1.56 3.125 1.56 50 1.56 9 3.125 6.25 6.25 3.125 50 3.125 10
3.125 3.125 25 6.25 50 3.125 *Multidrug-resistant bacterial strains
isolated from clinical specimens from patients in University
Hospital Olomouc: MRSA--methicillin-resistant Staphylococcus aureus
4591, Staphylococcus haemolyticus (a fluoroquinolone-resistant
strain) 16568, Enterococcus faecium (vancomycin-resistant strain)
VanA 419/ana, Staphylococcus epidermidis (methicilin-resistant
strain) 8700/B
[0119] In all cases, the value of the minimum inhibitory
concentration (MIC) which is the concentration of test substance in
the medium, which inhibited 100% of the growth of the tested
bacteria, was equal to the minimum bactericidal concentration (MBC)
which is the concentration at which 100% of the the tested bacteria
were killed. The MBC value was tested so that the bacteria tested
for MIC were inoculated into a medium, which did not contain an
inhibitor, and were monitored for growth.
[0120] Benefits of Lipophosphonoxins of the Second Generation:
[0121] Compared to LPPO of the first generation (J. Med. Chem.
2011, 54(22), 7884-7898, CZ PV 2011-312, EP2527351), the LPPO of
the second generation show a much broader spectrum of antibacterial
activity. Surprisingly, they are mainly effective against
clinically important gram-negative bacterial strains and against
harmful multiresistant bacterial strains occurring in the hospital
environment.
[0122] According to the OECD404 test for skin irritation in
rabbits, LPPO, specifically the compound of Example 3, is not an
irritant.
[0123] Maximum tolerated dose in mice was very high, for the
compound of Example 3 and oral administration the maximum tolerated
dose was 1500 mg/kg of bodyweight.
[0124] The mechanism of action of LPPO of the second generation
consists in the selective disruption of the bacterial cell
membrane.
[0125] LPPO are well soluble in water.
[0126] LPPO exhibit high stability at a wide pH range (1-8).
[0127] Resistance formation against LPPO is very unlikely, since
LPPO directly target the cell membrane, which is crucial for the
life of the bacteria.
INDUSTRIAL APPLICABILITY
[0128] As antibacterial agents, lipophosphonoxins of this invention
can be used as active ingredients of pharmaceutical compositions
for the treatment of even very resistant bacterial infections, as
ingredients of disinfectants and/or of selective culture media.
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